Partial characterization of a new adenovirus lineage discovered in testudinoid turtles

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Highlights

  • Novel adenoviruses (with balanced G + C) were detected in different turtles.

  • These viruses form a lineage different from every earlier adenovirus genus.

  • The novel virus lineage is hypothesized to have coevolved with testudinoid turtles.

  • Establishment of a new genus (with a putative name Testadenovirus) is proposed.

Abstract

In the USA and in Hungary, almost simultaneously, adenoviruses of a putative novel lineage were detected by PCR and sequencing in turtles belonging to four different species (including two subspecies) of the superfamily Testudinoidea. In the USA, partial sequence of the adenoviral DNA-dependent DNA polymerase was obtained from samples of a captive pancake tortoise (Malacochersus tornieri), four eastern box turtles (Terrapene carolina carolina) and two red-eared sliders (Trachemys scripta elegans). In Hungary, several individuals of the latter subspecies as well as some yellow-bellied sliders (T. scripta scripta) were found to harbor identical, or closely related, putative new adenoviruses. From numerous attempts to amplify any other genomic fragment by PCR, only a nested method was successful, in which a 476-bp fragment of the hexon gene could be obtained from several samples. In phylogeny reconstructions, based on either DNA polymerase or hexon partial sequences, the putative new adenoviruses formed a clade distinct from the five accepted genera of the family Adenoviridae. Three viral sub-clades corresponding to the three host genera (Malacochersus, Terrapene, Trachemys) were observed. Attempts to isolate the new adenoviruses on turtle heart (TH-1) cells were unsuccessful. Targeted PCR screening of live and dead specimens revealed a prevalence of approximately 25% in small shelter colonies of red-eared and yellow-bellied sliders in Hungary. The potential pathology of these viruses needs further investigation; clinically healthy sliders were found to shed the viral DNA in detectable amounts. Based on the phylogenetic distance, the new adenovirus lineage seems to merit the rank of a novel genus.

Introduction

Adenoviruses (AdVs) are common infectious agents in a large number of vertebrate animals worldwide. The family Adenoviridae is presently divided into five genera (Harrach et al., 2011). Mastadenovirus and Aviadenovirus were established long ago and currently contain exclusively mammalian or avian AdVs, respectively. The most recently approved genus, Ichtadenovirus, was established for the only known piscine AdV isolated from the white sturgeon (Benkő et al., 2002, Benkő and Doszpoly, 2011). Two additional genera consist of AdVs of mixed host origin. The first representatives of the genus Atadenovirus (Both, 2011) were found in ruminants and birds, and an atadenovirus was also demonstrated in a marsupial host (Thomson et al., 2002). The name of the genus reflects the strikingly high A + T content observed in the genome of the first characterized atadenoviruses (Benkő and Harrach, 1998). Because of their large phylogenetic distance from the avian and mammalian AdVs, this genus had been hypothesized to have co-evolved with some lower vertebrates, most probably with reptiles (Harrach, 2000). This hypothesis was supported by the first genome-wide analysis of a reptilian AdV (snake adenovirus 1), revealing the presence of numerous genus–specific genes and ORFs (Davison et al., 2003). Interestingly however, the nucleotide (nt) composition of the SnAdV-1 genome (50.21% G + C) was not biased (Farkas et al., 2008). Development of a nested PCR with consensus primers, targeting the DNA-dependent DNA polymerase gene, enabled the recognition of numerous novel atadenoviruses in many additional squamate hosts (snakes and lizards) (Wellehan et al., 2004, Papp et al., 2009, Hyndman and Shilton, 2011, Abbas et al., 2011). Based on the sequence of shorter or longer genome parts, the G + C content of every atadenovirus of squamate origin examined to date was found to be non-biased. This notion prompted a hypothesis on the correlation between A + T richness and assumed occasional host switches of adenoviruses (Benkő and Harrach, 2003). The fifth genus, Siadenovirus, initially contained the only frog AdV isolate (FrAdV-1) together with turkey adenovirus 3, an exceptional avian AdV known for its broad host spectrum and strong pathogenicity (Davison et al., 2000, Beach et al., 2009). While it had been hypothesized that siadenoviruses had co-evolved with amphibians (Benkő and Harrach, 2003, Davison et al., 2003), this was seriously challenged by the high A + T content of every known siadenovirus and by the discovery of further new siadenoviruses in birds of different species (Zsivanovits et al., 2006, Katoh et al., 2009, Wellehan et al., 2009, Kovács et al., 2010, Park et al., 2012), as well as in Sulawesi tortoises (Rivera et al., 2009). Birds are known to be infected by aviadenoviruses, atadenoviruses, and siadenoviruses. The detection of a siadenovirus in Sulawesi tortoises indicated that besides birds and ruminants, some reptiles might also harbor AdVs from diverse genera.

From 2007 on, hitherto unseen AdVs, representing clearly a separate lineage that most probably merits the status of a new genus, were detected by PCR and sequencing in a number of turtles belonging to several species of the superfamily Testudinoidea (Turtle Taxonomy Working Group, 2011). In the present work, attempts to characterize this new AdV lineage and the results of a pilot study aiming at the assessment of the prevalence in some small pond slider populations in Hungary are described.

Section snippets

Origin and processing of the samples

The first specimens found, almost simultaneously in the USA and Hungary, to contain the DNA of a new virus, were from a captive red-eared (Trachemys scripta elegans) and a dead yellow-bellied (Trachemys scripta scripta) slider, respectively. Subsequently, further closely related AdV sequences were detected in four eastern box turtles (Terrapene carolina carolina) and in a pancake tortoise (Malacochersus tornieri) in the USA.

For preliminary assessment of the prevalence of the newly recognized

PCR screenings

Novel turtle adenoviruses were discovered during routine nested-PCR screening of animal samples and carcasses for the presence of AdVs in Hungary and in the USA in 2007. The first positive samples were from a small, young (<1 year) yellow-bellied slider that had died in a pet shop in Budapest, and from a red-eared slider cloacal swab submitted to the College of Veterinary Medicine at the University of Florida, Gainesville while the animal was in quarantine at a zoological facility. Direct

Discussion

Because of their wide host range, AdVs make an interesting model for the study of host-virus coevolution (Benkő and Harrach, 2003). According to the earliest hypothesis, the five recognized groups of AdVs had been supposed to represent the AdV lineages each co-speciating with what were then considered to be the major vertebrate classes (Benkő and Harrach, 2003, Davison et al., 2003). While the original observation leading to this conclusion was made on the basis of the results of phylogenetic

Acknowledgements

This work was supported by the Hungarian Scientific Research Fund (OTKA Grant K72484). The authors are grateful to Rachel Marschang and Tibor Papp for their help in the virus isolation attempts, as well as to Attila Puskás for providing a sample.

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